WO2015133800A1

WO2015133800A1 - Marker for predicting anticancer therapeutic reaction with respect to egfr tyrosine kinase inhibitor

Info

Publication number: WO2015133800A1
Application number: PCT/KR2015/002054
Authority: WO
Inventors: 조병철; 김한상
Original assignee: 연세대학교 산학협력단
Priority date: 2014-03-04
Filing date: 2015-03-03
Publication date: 2015-09-11
Also published as: KR20150104264A; KR101622454B1

Abstract

The present invention provides a method for providing information for predicting the prognosis of cancer therapy, and a composition and a kit for predicting the prognosis of cancer therapy using an EGFR tyrosine kinase inhibitor. The present invention involves measuring the level of expression of a gene or a protein selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2, thereby making it possible to predict the prognosis of cancer therapy using an EGFR tyrosine kinase inhibitor, and hence the present invention can be used to advantage in order to select a drug for treating patients.

Description

Anticancer Response Prediction Markers for EGFR Tyrosine Kinase Inhibitors

The present invention relates to anticancer treatment response predictive markers for EGFR tyrosine kinase inhibitors.

Head and neck cancer is the sixth leading cause of cancer deaths worldwide, with 650,000 new cases diagnosed each year. About 60% of patients with squamous cell head and neck cancer will receive a locally advanced, multidisciplinary treatment that cannot be resected at diagnosis. In multi-faceted care, the majority (70%) of patients have local or local recurrence. In 10% of patients at diagnosis, distant metastasis is found. Most recurrent or metastatic disease will receive a single chemotherapy, combined chemotherapy or targeted therapy.

Epidermal growth factor receptor (EGFR) is frequently overexpressed in squamous cell head and neck cancer, and is associated with a poor prognosis. Several other studies have known the relationship between EGFR activity signaling and tumor survival.

Targeted therapeutic agents, EGFR inhibitors, are largely divided into monoclonal antibodies and small molecule EGFR tyrosine kinase inhibitors. The monoclonal antibody erbitux® (Cetuximab) is widely used for the treatment of metastatic head and neck cancer under US FDA approval, and is a small molecule EGFR tyrosine kinase inhibitor, gefitinib, erlotinib, and daco. Nitinib and afatinib are currently in clinical studies in patients with metastatic head and neck cancer.

Among the EGFR target antibody therapeutics, cetuximab, in particular, has shown clinical anticancer effects in squamous cell head and neck cancer. The addition of cetuximab to 5FU and platinum as a primary drug resulted in a median overall survival of 10.1 months compared to 7.4 months when only 5FU and platinum were used.

However, although EGFR is known to be an important therapeutic target for head and neck cancer, the general drug response rate is known to be 10-15%, which is significantly lower than that of EGFR-targeted drugs in lung cancer. The reason for this is that it is not known in which patient group the drug response is good, and therefore, there is a need for the discovery of biomarkers to select a patient group that can maximize the effect on the EGFR tyrosine kinase inhibitor.

It is an object of the present invention to provide prognostic predictive markers for the cancer therapeutic effects of EGFR tyrosine kinase inhibitors.

In order to achieve the above object, the present invention was to find a gene group that differs between the drug response group and the drug non-response group for EGFR tyrosine kinase inhibitor by measuring gene expression in tumor tissue of head and neck cancer patients.

As a result, in the present invention, it was confirmed that the gene expression of the inflammatory cytokines IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and / or PTGS2 was highly expressed in the non-drug group for EGFR tyrosine kinase inhibitor. . Therefore, when the gene or protein expression level is high in the patient's sample for predicting prognosis, the prognosis of cancer treatment by the EGFR tyrosine kinase inhibitor is predicted to be poor, and this is used for diagnosing the cancer prognosis of the patient or selecting an anticancer agent. It can be used as information.

Thus, the present invention

Measuring at least one gene or protein expression level selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2 from biological samples isolated from cancer patients;

Comparing the gene or protein expression level with the gene or protein expression level in the drug response group for the EGFR tyrosine kinase inhibitor; And

Determining that the prognosis of cancer treatment by the EGFR tyrosine kinase inhibitor is poor when the gene or protein expression level is higher than the gene or protein expression level in the drug response group to the EGFR tyrosine kinase inhibitor.

Provided are informational methods for predicting the prognosis of cancer treatment with EGFR tyrosine kinase inhibitors.

In the present invention, the "pharmaceutical response group" means a patient with a progression-free survival for a drug longer than a certain period, the patient with a progression-free survival is less than the period is referred to as a "pharmaceutical non-response group" It is called. The period of dividing the drug response group and the drug non-response group may vary depending on the type of drug, the severity and condition of the disease, and the like. For example, the drug response group has a period of 3 months, 4 months, 5 months, and 6 months. And drug non-responders. In one embodiment of the present invention divided the drug response group and the drug non-response group about 4 months before the progression-free survival period.

Determining that the expression level of the gene or protein is high when compared with the drug response group means that the statistical analysis is significantly higher than the mean expression level in the drug response group.

In the present invention, EGFR tyrosine kinase when the gene or protein expression level of one or more of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2 is higher than the gene or protein expression level in the drug response group for EGFR tyrosine kinase inhibitor. There is a high likelihood of drug resistance to inhibitors, which can be predicted to be a poor prognosis for cancer treatment. In the following examples, tumor tissues of cancer patients were compared with their gene levels using samples. However, since these biomarkers are detectable in blood, protein levels can also be compared.

In the present invention, a biological sample isolated from a cancer patient may include tumor tissue of a cancer patient, cells or nucleic acids separated therefrom; Blood, serum, plasma or proteins isolated therefrom.

The method for measuring the expression level of a gene or protein in the above method may be carried out including a known process for separating mRNA or protein from a biological sample using known techniques. Determination of the expression level of the gene is preferably to measure the level of mRNA, the determination of the expression level of the gene can be carried out through various methods known in the art. For example, RT-PCR (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine , 102-108, CRC press), hybridization reaction using cDNA microarray (Sambrook et al., Molecular Cloning.A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization reaction (Sambrook et al. , Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)).

For example, when performed according to the RT-PCR protocol, first, total RNA is isolated from cells treated with a sample, and then single-stranded cDNA is prepared using oligo dT primers and reverse transcriptase. Then, single-stranded cDNA is used as a template, and PCR reaction is performed using a gene-specific primer set. Then, PCR amplification products can be electrophoresed, and the formed bands can be analyzed to measure changes in expression levels of genes.

In addition, protein expression levels can be measured through various immunoassay methods known in the art. For example, but not limited to radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbentassay (ELISA), capture-ELISA, sandwich analysis. The immunoassay or method of immunostaining is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999. In the present invention, the cancer may be head and neck cancer, in particular squamous head and neck cancer.

Meanwhile, the EGFR tyrosine kinase inhibitor may be selected from gefitinib, erlotinib, daconitinib, and afatinib. In one embodiment of the present invention, the gene expression level of proinflammatory cytokines such as IL6, IL8, PLA2G2A, PTGS2, etc., has been shown to predict the prognosis of cancer treatment by daconitinib, but the type of EGFR tyrosine kinase inhibitor Is not limited thereto.

In one embodiment, the method identifies mutations in at least one gene selected from the group consisting of PIK3CA and PTEN from a biological sample isolated from a cancer patient and, if present, modulates the treatment of cancer with an EGFR tyrosine kinase inhibitor. The prognosis may further include providing information as having a poor prognosis. As can be seen in the following examples, the presence of PI3K pathway mutations, ie PIK3CA or PTEN mutation, together with the expression level of the proinflammatory cytokine, the prognostic prediction of cancer treatment by EGFR tyrosine kinase inhibitor More accurate (see FIG. 3).

The invention also predicts the prognosis of cancer treatment by an EGFR tyrosine kinase inhibitor comprising a probe or primer having a sequence complementary to one or more genes selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2. It provides a composition for.

The composition is used to predict the prognosis of cancer treatment by confirming the expression level of the genes, the composition according to the present invention will comprise a probe or primer having a sequence complementary to the gene.

The term “probe” refers to a linear oligomer of natural or modified monomers or linkages, including deoxyribonucleotides and ribonucleotides, capable of specific hybridization to a target nucleotide sequence, either naturally occurring or artificially It is synthesized. Probes of the invention are preferably single chain and oligodioxyribonucleotides.

The term “primer” refers to a single-stranded oligonucleotide that can act as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) at a suitable temperature. it means. Suitable lengths of primers are typically 15-30 nucleotides, although varying with various factors, such as temperature and the use of the primer. Short primer molecules generally require lower temperatures to form hybrid complexes that are sufficiently stable with the template.

The sequence of the primer does not need to have a sequence that is completely complementary to some sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to perform the primer-specific function. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the nucleotide sequence that is a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the gene sequence to act as a primer. The design of such primers can be easily carried out by those skilled in the art with reference to the above-described nucleotide sequence, for example, by using a program for primer design (eg, PRIMER 3 program).

The present invention also provides a composition for predicting the prognosis of cancer treatment by EGFR tyrosine kinase inhibitors comprising an antibody that specifically binds to at least one protein selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2. to provide.

The composition is used to predict the prognosis of cancer treatment by confirming the expression level of the proteins, and the composition according to the present invention includes an antibody that specifically binds to the proteins.

Antibodies specific for the proteins may include polyclonal antibodies, monoclonal antibodies, human antibodies and humanized antibodies. Examples of the antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments. ; Diabody; Linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995)); single chain antibody molecules; And multispecific antibodies formed from antibody fragments and the like.

Methods of preparing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be prepared by injecting the mammal with one or more immunizing agents, if necessary, with an adjuvant. Typically, the immunizing agent and / or adjuvant is injected into the mammal several times by subcutaneous injection or intraperitoneal injection. Antibodies can be prepared by the hybridoma method described in Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (see, eg, US Pat. No. 4,816,576). have.

Monoclonal antibodies are also described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991). It can be isolated from phage antibody libraries using the techniques described. Monoclonal antibodies in the present invention are specifically those in which a portion of the heavy and / or light chain is identical to the corresponding sequence of an antibody derived from a particular species or an antibody belonging to a particular antibody class or subclass, provided that it exerts the desired activity or Although homologous, the remainder of the chain (s) includes "chimeric" antibodies that are identical or homologous to antibodies from other species or to antibodies belonging to different antibody classes or subclasses or fragments of such antibodies (Morrison et. al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

The composition for predicting prognosis of cancer treatment according to the present invention may be included in the form of a kit.

The kit may comprise probes, primers or antibodies capable of measuring the level or amount of one or more genes or protein expression levels or proteins selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2, and these The definition of is as described above.

Optionally, when the kit is subjected to a PCR amplification process, reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Thermally stable DNA polymerases obtained from Pyrococcus furiosus (Pfu), DNA polymerase cofactors and dNTPs, and when the kits are subjected to an immunoassay, the kits of the invention may optionally contain secondary antibodies and labels It may include a substrate. Furthermore, the kits according to the invention can be produced in a number of separate packaging or compartments comprising the reagent components described above.

In addition, the composition for predicting prognosis of cancer treatment of the present invention may be included in the form of a microarray.

In the microarray of the present invention, a probe, primer, or antibody capable of measuring the expression level of the gene or protein is used as a hybridizable array element and immobilized on a substrate. Preferred substrates may include suitable rigid or semi-rigid supports, such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. have. The hybridization array element is arranged and immobilized on the substrate, and such immobilization can be performed by a chemical bonding method or a covalent binding method such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element can be coupled to the substrate via a linker (eg, ethylene glycol oligomer and diamine).

On the other hand, when the sample applied to the microarray of the present invention is a nucleic acid can be labeled, it can be hybridized with the array element on the microarray. Hybridization conditions may vary, and detection and analysis of the degree of hybridization may vary depending on the labeling substance.

Information for predicting the prognosis of cancer treatment by the EGFR tyrosine kinase inhibitor obtained by using the composition for predicting the prognosis of the cancer treatment to investigate the expression level of proinflammatory cytokines is PI3K pathway mutations, namely PIK3CA and PTEN. Analysis with information on whether one or more genes selected from the group consisting of mutations can be used to more accurately predict the prognosis of cancer treatment by EGFR tyrosine kinase inhibitors.

According to the present invention, by measuring the expression level of one or more genes or proteins selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2, the prognosis of cancer treatment by EGFR tyrosine kinase inhibitors can be predicted. It can be usefully used for the selection of a medicament for treatment.

1A to 1G show gene expression values corresponding to proinflammatory cytokines in drug response group (S) and non-response group (R) based on 4 months of progression free survival (PFS). (IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2, respectively).

Figure 2 is a heat map of the gene expression corresponding to inflammatory cytokines between drug response group (S) and non-response group (R) based on 4 months progression-free survival.

3A shows progression free survival (PFS) and survival (OS) for all patients (n = 48), and FIG. 3B shows progression free survival (PFS) and survival (OS) according to PIK3CA / PTEN / AKT1 mutation status. (n = 33), FIG. 3C shows progression-free survival (PFS) and survival (OS) (n = 20) according to inflammatory cytokine expression, and FIG. 3D shows PIK3CA / PTEN / AKT1 mutation status and inflammation. Kaplan-Meier estimates of progression-free survival (OS) and survival (OS) (n = 33) for sex cytokine expression, respectively.

In order to find markers associated with therapeutic prognosis following the use of EGFR tyrosine kinase inhibitors in head and neck cancer, we performed expression of several genes by applying the NanoString® method.

In this study, 48 patients with recurrent / metastatic squamous head and neck cancer who received EGFR tyrosine kinase inhibitor dacomitinib were enrolled in this study. They had a disease-free survival of 3.9 months (PFS) and an overall survival (OS) of 8.2 months, with 12 tumor tissues available for gene expression analysis. In the present study, the data of FIGS. 1 and 2 were divided into drug-response group and drug-less-responder group when the disease free survival period was more than 4 months, but the experimenter did not know the clinical data. Proceeded.

(Extraction of mRNA from Tumor Tissue)

MRNA was extracted from the 12 tumor tissues for gene expression analysis. Concentration was measured by quantitative analysis and mRNA analysis using bioanalyzer was performed by qualitative analysis, and gene expression was measured by the sample that passed both qualitative and quantitative analysis.

(Measurement and analysis of gene expression using nanostring)

NanoString® Technologies ( http://www.nanostring.com/) is a technology introduced in Nat Biotechnol. 2008 Mar; 26 (3): 317-25 in 2008. It is a technique that can quantitatively measure gene expression.

First, the mRNA extracted from the sample tissue and a capture probe were hybridized, and only the hybridized mRNA-capture probe molecule was selected and inserted into the data analyzer, and the gene expression value was quantitatively measured. Six fluorescent substances are attached to the ends of the capture probes, and a combination thereof can be used to quantitatively measure the expression value of a desired gene.

(Analysis of Gene Expression)

As a result of measuring gene expression through a nanostring (NanoString®) method, the results shown in Table 1 were obtained.

Table 1

	Non-response group 1	Non-response group 2	Non-response group 3	Unresponsive group 4	Non-response group 5	Unresponsive Group 6	Non-response group 7	Reaction group 1	Reaction group 2	Reaction Group 3	Reaction Group 4	Reaction Group 5
IL1A	98.29	262.74	196.34	140.79	101.49	101.3	22.56	12.13	6.75	71.21	69.02	9.09
TNF	33.46	62.2	100.69	70.83	82.09	82.3	75.21	20.04	28.35	44.15	113.38	25.67
IL8	707.38	1677.26	503.45	197.8	252.23	373.53	185.01	93.86	93.17	158.09	36.97	48.66
IL6	417.21	146.92	105.72	2.59	5.97	56.98	73.7	1.05	2.03	52.7	2.46	2.67
PTGS2	241.02	4512.73	213.96	70.83	123.88	322.88	189.53	11.07	14.85	266.33	27.11	8.02
MMP3	151.62	229.5	178.72	109.7	122.38	240.58	43.62	7.38	8.78	121.06	39.44	13.9
PLA2G2A	1326.39	561.95	244.17	99.33	594	645.76	2451.8	45.35	15.53	431.54	69.02	96.79

Table 1 is a gene expression value is expressed to have a positive value from 0 to 5000, the higher the value indicates that the expression is higher.

As shown in FIG. 1 and FIG. 2, the data were analyzed. As shown in FIG. 1, gene expressions such as IL6, IL8, PLA2G2A, IL1A, TNF, MMP3, and PTGS2 resulted in drug response groups (no There was a difference between the disease period of 4 months or more) and the non-responder group (less than 4 months of the disease-free period).

To show this as a picture, the heatmap of FIG. 2 shows that the gene expression of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2 is highly expressed in the non-responsive group to daconitinib. there was.

In particular, IL6, IL8, PLA2G2A, IL1A, TNF, and PTGS2 are pro-inflammatory cytokines, and in this study, there was a correlation between drug resistance and pro-inflammatory cytokine. .

Inflammatory cytokines have been reported to affect tumor formation, growth and metastasis (Klampfer L. Cytokines, inflammation and colon cancer. Current Cancer Drug Targets. 2011, 11, 451-464.) To date, there have been no reports of an increase in proinflammatory cytokines in the peri-tumor or serum of EGFR inhibitors. In this study, it was confirmed that an increase in inflammatory cytokines in the tumor region or serum may increase resistance to anticancer drugs. This seems to be based on the increase in proinflammatory cytokines that enhance the survival signal of tumor cells and weaken the apoptosis signal. Thus, increased expression of proinflammatory cytokines can be utilized as useful biomarkers for predicting the anticancer efficacy of EGFR inhibitors.

(Relevance with PI3K Path Variation)

In 20 patients whose data on both proinflammatory cytokine expression and PI3K pathway mutations were obtained, there was no correlation between proinflammatory cytokine expression and PI3K pathway mutations.

However, as can be seen in FIG. 3, when both the PI3K pathway mutation and the inflammatory cytokine expression were confirmed, statistically significant results were obtained, and as a result, the accuracy of predicting cancer prognosis was increased. It was confirmed that it can.

[FIG. 3A] shows disease free survival (PFS) and survival (OS) for all patients (n = 48). [FIG. 3B shows disease free survival (PFS) and PIK3CA / PTEN / AKT1 mutation status. Survival (OS), [FIG. 3C] shows disease-free survival (PFS) and survival (OS) according to inflammatory cytokine expression, [FIG. 3D] PIK3CA / PTEN / AKT1 mutation status and inflammatory cytokines Kaplan-Meier estimates of disease free survival (OS) and survival (OS) according to the expression of Cain are presented.

It can be confirmed through p value that the statistical significance of [FIG. 3d] is higher than that of [FIG. 3b] and [FIG. 3c].

Claims

Measuring at least one gene or protein expression level selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2 from biological samples isolated from cancer patients;

Comparing the gene or protein expression level with the gene or protein expression level in the drug response group for the EGFR tyrosine kinase inhibitor; And

EGFR tyrosine comprising providing information that the prognosis of cancer treatment with an EGFR tyrosine kinase inhibitor is poor when the gene or protein expression level is higher than the gene or protein expression level in a drug response group to the EGFR tyrosine kinase inhibitor. Informational method for predicting the prognosis of cancer treatment by kinase inhibitors.
The method of claim 1,

The cancer is head and neck cancer information providing method.
The method of claim 1,

And said cancer is squamous head and neck cancer.
The method of claim 1,

The EGFR tyrosine kinase inhibitor is selected from gefitinib (erfitinib), erlotinib (erlotinib), daconitinib (dacomitinib) and apatinib (afatinib).
The method of claim 1,

The method further

Identifying whether one or more genes selected from the group consisting of PIK3CA and PTEN are mutated from biological samples isolated from cancer patients and providing information as a poor prognosis for cancer treatment with EGFR tyrosine kinase inhibitors when the mutation is present. Informational method comprising the steps.
A composition for predicting prognosis of cancer treatment by an EGFR tyrosine kinase inhibitor comprising a probe or primer having a sequence complementary to at least one gene selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3, and PTGS2.
The method of claim 6,

The cancer is a composition for predicting the prognosis of cancer treatment by EGFR tyrosine kinase inhibitor that is head and neck cancer.
The method of claim 6,

The cancer is a composition for predicting prognosis of cancer treatment by EGFR tyrosine kinase inhibitors that are squamous head and neck cancer.
The method of claim 6,

The EGFR tyrosine kinase inhibitor is for predicting the prognosis of cancer treatment by the EGFR tyrosine kinase inhibitor is selected from gefitinib, erlotinib, dacomitinib and apatinib. Composition.
A composition for predicting prognosis of cancer treatment by an EGFR tyrosine kinase inhibitor comprising an antibody that specifically binds at least one protein selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3, and PTGS2.
The method of claim 10,

The cancer is a composition for predicting the prognosis of cancer treatment by EGFR tyrosine kinase inhibitor that is head and neck cancer.
The method of claim 10,

The cancer is a composition for predicting prognosis of cancer treatment by EGFR tyrosine kinase inhibitors that are squamous head and neck cancer.
The method of claim 10,

The EGFR tyrosine kinase inhibitor is for predicting the prognosis of cancer treatment by the EGFR tyrosine kinase inhibitor is selected from gefitinib, erlotinib, dacomitinib and apatinib. Composition.